PROJECT SUMMARY Nuclear receptors are a superfamily of ligand-regulated transcription factors that play fundamental roles in human development, homeostasis, and disease. These receptors interact with small molecules, protein partners, and DNA sequences to regulate the transcription of specific target genes. Developing ligands that stabilize specific conformational states of the receptor, and thus drive the transcription of specific target genes, is a prerequisite for developing effective nuclear receptor therapeutics. The nuclear receptor Nurr1 (NR4A2) is widely recognized as a therapeutic target for Parkinson's disease, potentially modifying both the symptoms and progression of the disease. Current therapeutics for Parkinson's disease are symptom-modifying only and lose efficacy as the disease progresses. Although ?Nurr1 agonists? have been reported in both the scientific and patent literature, there is little evidence these ligands directly activate the receptor. The only published crystal structure of Nurr1 reveals two distinctive features that have hindered progress developing small molecules targeting this receptor: Nurr1 lacks both the canonical nuclear receptor ligand binding pocket and the classical binding site for protein partners. Using an orthogonal drug development strategy called disulfide-trapping, we identified ~50 small molecules that bind directly to Nurr1 and form covalent adducts with a native cysteine residue in the ligand binding domain. We also identified an endogenous ligand that forms a reversible covalent adduct with the same cysteine residue. Co-crystal structures for three of these ligand-receptor complexes show Nurr1 in three distinctly different conformations. The proposed research will capitalize on these findings to develop chemical probes for Nurr1 that can be used to unravel the receptor's complex biology. Successful completion of these aims will define the relationships between individual ligand scaffolds, Nurr1 conformational states, and specific Nurr1 target genes, thereby providing the foundation for rationally developing new PD therapeutics. In Aim 1, we will generate covalent ligands for Nurr1, suitable for cellular assays, and use them to identify the target genes associated with different conformational states of the receptor. In Aim 2, we will identify functional analogs of the endogenous Nurr1 ligand that will enable cellular studies probing the receptor's regulation. In Aim 3, we will identify ligands that stabilize additional conformations of the receptor (e.g. heterodimer with RXR) and solve co-crystal structures of the resulting Nurr1 complexes.